Method for forming molecular layers having high density of primary amine group on solid supports

ABSTRACT

Provided is a method for forming solid substrates having high density of primary amine group on its surface, in which the primary amine groups on the surface of an aminosilylated substrate are treated with aziridine or an aziridine derivative. The surface density of the primary amine functional groups (—NH 2 ) on the very top surface of a substrate can be drastically increased.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a method for forming molecularlayers having high density of primary amine groups on solid supports.

More particularly, this invention relates to a method for forming eitherlinear or hyper-branched polymer layers having high density of primaryamine groups on hydroxylated solid supports. Also, this inventionrelates to a method for growing polymer layers having high density ofprimary amine group on the solid substrates while utilizing the primaryamine on the surface of an aminosilylated substrate to initiate thering-opening polymerization of aziridine or aziridine derivatives.

2. Description of the Related Art

Silylation, particularly aminosilylation of the surface on solidsupports has been applied in a variety of fields includingimmobilization of biomolecules such as enzyme and antibody,immobilization of inorganic catalysts, modification of electrodes,chromatography, and formation of a self-assembled building foundationusing various kinds of molecules such as ionic polymer, nonlinearoptical chromophore, fullerene, porphyrin, transition metal complex andinorganic colloidal particle.

The physico-chemical characteristics of the aminosilane layer formed onthe surface of solid supports are very important because those determinethe structures and the final functionality of the thin film whileinfluencing the shape and the surface density of the immobilized or selfassembled molecule.

On the other hand, polymerization initiated on the surface of asubstrate has attracted such big attention due to its wideapplicability. This invention suggests the fact that new molecularstructure accompanying the polymerization can modify the surfaceproperties of a support, and can be chosen to meet various needs.Moreover, chemical bonding between the surface-bound initiator on asupport and polymer chain guarantees the excellent stability.

In addition, a “grafting from” methodology will provide desirablemorphologies on the surface of a support. Therefore, it seems naturalthat people are interested in polymerization initiated on solidsupports.

Ulman et al. showed that 2-ethyl-2-oxazoline can be cationicallypolymerized on a gold surface modified with -hydroxyl thiol (J. Am.Chem. Soc. 120, 243(1998)), and they revealed that surface-initiatedanionic polymerization of styrene is advantageous over “grafting onto”technique (J. Am. Chem. Soc. 121, 1016(1999)). Recently, Grubbs et al.successfully demonstrated that ring-opening metathesis polymerization(ROMP) catalyst polymerizes norbornene at a solid support to make apolymer brush successfully (J. Am. Chem. Soc. 121, 4088(1999)).

It has been generally known that the surface density of the primaryamine functional group (—NH2) is about 3.5 amines/100 Å² on the very topsurface when a relevant reagent was applied on the surface of a solidsupport. However, the conventional solid supports having such a lowdensity of primary amine group suffer from limited applicability andtherefore need to be improved. A solid support having primary aminegroups on its surface is suitable as a substrate for DNA chip or variouskinds of biochip. However, the density of 3.5 amines per 100 Å² on thesurface of a support is not high enough to hold hydrogels containing DNAoligonucleotides or other biomolecules of different effects becausethose microbeads need stronger adhesion for the higher reusability.Therefore, the conventional solid supports have not served for the highstability of the biochips and the wide applicability.

SUMMARY OF THE INVENTION

The present invention is directed to providing a method for formingmolecular layers with high density of primary amine groups onsubstrates.

The present invention is related to a method for forming molecularlayers with high density of primary amine group on solid supports byallowing the primary amine groups on the surface of an aminosilylatedsubstrate to react with aziridine or aziridine derivatives.

This invention is also related to aminosilylated substrates having highdensity of primary amine groups, generated according to the method ofthe invention.

The invention is characterized by treating primary amine groups ofaminosilylated substrates with aziridine or its derivatives therebytailoring the chemical and physical properties of the thin films on thesubstrate. It is preferable to use aziridine of formula 1

or a protected aziridine derivative of formula 2

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention, and many of the attendantadvantages thereof, will be readily apparent as the same becomes betterunderstood by the reference to the following detailed description whenconsidered in conjunction with the accompanying drawings, wherein:

FIG. 1 shows the reaction process between aziridine and primary aminegroups on the surface of an aminosilylated substrate where aring-opening polymerization occurs to form poly(ethyleneimine) chains(linear or branched) on the surface.

FIG. 2 shows a stepwise growth of poly (ethyleneimine) by reacting aprotected aziridine, benzyl 1-aziridinecarboxylate (N—Cbz aziridine),with the primary amine groups on the surface of an aminosilylatedsubstrate.

FIG. 3 is a graph showing UV-vis spectra of a4-nitrobenzaldimine-derivatized surface of an aminosilylated substratebefore and after the polymerization according to Example 1.

FIG. 4a is an atomic force microscope (AFM) image showing the surfacemorphology of an aminosilylated (non-polymerized) substrate preparedaccording to Example 1.

FIG. 4b is an atomic force microscope (AFM) image showing the surfacemorphology of a polymerized, aminosilylated substrate prepared accordingto Example 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is characterized by treating primary amine groups ofaminosilylated substrates with aziridine or its derivatives therebytailoring the chemical and physical properties of the thin films formedon the substrate. In one embodiment, it is preferable to use aziridineof formula 1

In another embodiment, it is preferable to use a protected aziridinederivative. Any aziridine compound in which the nitrogen functionalgroup is protected can be used as the protected aziridine derivative. Apreferred protected aziridine compound is an aziridine derivative offormula 2.

The compounds of formula 2, wherein X is selected from the groupconsisting of hydrogen, nitro, alkoxy, and mixtures thereof arepreferable owing to the easy removal (i.e., deprotection) of theprotecting group after the polymerization reaction.

The primary amine groups in aminosilylated molecular layers andaziridine or aziridine derivatives are reacted by immersing (i.e.,contacting) the aminosilylated substrate in a solvent solution (e.g.,dichloromethane) containing the dissolved aziridine or aziridinederivatives and a catalytic amount of an acid (e.g., acetic acid). Thesolution is preferably heated under an inert atmosphere to increase thereaction rate (e.g., to reflux for 24 hours under nitrogen).

Due to the reactivity between aziridine and primary amines, aring-opening polymerization occurs which generates poly(ethyleneimine)in the form of either a linear chain or a branched chain on the surfaceof the aminosilylated substrate (see FIG. 1). When using the protectedaziridines, the ring-opening reaction produces a linear chain withoutforming branches on the surface of an aminosilylated substrate, in whicha deprotection step should follow the ring-opening step for the furthergrowth.

In this invention, a method for forming molecular layers with highdensity of primary amine groups on the surface of aminosilylatedsubstrates will be explained in more detail with the reference tofigures.

First, a solid support with clean and dry surface is immersed in(contacted with) a mixture including an aminosilane compound and asuitable solvent for an appropriate reaction time to form the reactiveprimary amine functional groups on the surface of the support. Thesupport is preferably a hydroxylated support, such as a silicon wafer,fused silica, and other like materials known in the field. Theaminosilane compound to be used preferably does not form acidicby-products. Preferred aminosilane compound are(3-aminopropyl)diethoxymethylsilane,(3-aminopropyl)ethoxydimethylsilane, (3-aminopropyl)triethoxysilane, andmixtures thereof. A suitable solvent is any solvent that can be used todissolve the aminosilane compound, which include organic solvents suchas toluene and benzene. The resulting aminosilylated substrate isthoroughly washed with the solvent and dried in vacuum at roomtemperature.

The aminosilylated substrate is immersed in (contacted with) thesolution comprising the aziridine or aziridine derivative, the acidcatalyst, and the solvent. As described above, the solution ispreferably heated to reflux under the inert gas atmosphere. Anappropriate heating temperature of the reaction between theaminosilylated surface and the aziridine compound is preferably between40° C. and 100° C. The aziridine or aziridine derivatives will bevaporized over 100° C. and thus decreasing the efficiency of thereaction. The reactivity of the components will also decrease under 40°C. Acid catalysts to be used in the present invention include aceticacid, p-toluenesulfonic acid, and the like. Solvents to be used in thepresent invention are organic solvents such as dichloromethane, toluene,acetonitrile, and the like. Subsequently, the resulting substrate isthoroughly washed with the proper organic solvent.

When a protected aziridine is employed, a deprotection step to eliminatethe protecting group from the surface of the substrates should followthe reaction of the aminosilylated substrate with the protectedaziridine. For example, when benzyl 1-aziridinecarboxylate (or N-Cbzaziridine) is used as the protected aziridine, the substrate is immersedin neat triflouroacetic acid and sonicated at room temperature for thedeprotection. Subsequently, the surface of the substrate is washed witha copious amount of a solvent such as methanol to remove triflouroaceticacid remaining from the previous step. A particular acid such astrifluoroacetic acid, aqueous trifluoroacetic acid, and boron tribromide(BBr₃) can be used for the deprotection, and the best result can beobtained with neat trifluoroacetic acid (No other generic acids work forit).

In this invention, the substrates having molecular layers of thestructures shown in FIG. 1 and FIG. 2 are obtained. Referring to FIG. 1,ring-opening polymerization occurs to form a hyperbranched polymer onthe aminosilylated substrate by using aziridine. The polymerization canresult in either a linear chain or a branched chain depending on whetherthe primary amine is converted to a secondary or tertiary amine whenreacting with aziridine. In the latter case, the branching occurs whenthe nitrogen atom of the aminosilane compound or of the reactedaziridine reacts with more than one aziridine forming a hyperbranchedpoly (ethyleneimine) on the surface.

A reliable way to judge whether the branching occurs is to ascertain thesurface density of the end group (i.e., the primary amine). Absorbanceat 284 nm owing to 4-nitrobenzaldimine occurs when the primary amine isconverted to an imine using 4-nitrobenzaldehyde as described in Moon etal., Langmuir 13,4305 (1997), which in incorporated herein by reference.Thus, a sharp increase in absorbance is expected due to the increasednumber of primary amines after the polymerization. The absolute densityof the primary amine groups is also determined by hydrolyzing the iminegroups with a known amount of water as described in Moon et al.

Before treating with aziridine or the aziridine derivative, the surfaceof aminosilylated substrate has the comparatively flat structure with aconstant height and density. However, the growth accompanying branchingusually forms a rough surface due to the formation of irregular polymerchain lengths, which is believed to be caused by aziridine reactingrandomly with other aziridine molecules. Branching thus provides ahigher surface density of primary amines than obtained with a populationof similar chain lengths.

In order to prepare a better-defined (i.e., smoother) polymer layer onthe surface of the substrate, a stepwise polymerization is used.Referring to FIG. 2, the primary amines on the surface of anaminosilylated substrate are reacted with N-Cbz aziridine, providingonly modified amines on the very top surface of the substrate andsimilar polymers chain lengths as a result. This avoids the formation ofirregular polymer chain lengths that leads to a rougher polymer surface(i.e., a more uneven surface morphology).

In another embodiment of this invention, a solid support having a highdensity of primary amines is provided. An absolute surface density ofthe primary amines of at least greater than 3.5 amines/nm² ispreferable. A surface density of at least 10 amines/nm², with at least48 amines/nm², or greater are also obtainable.

Solid supports with a high surface density of primary amine groups inthis invention are highly useful in the development of DNA chips(arrays) or various others kinds of biochips. Descriptively, in the caseof DNA chip, hydrogels or polymers containing an oligonucleotide as wellas —N═C═O groups available for the chemical bonding with a primary aminecan be more stably attached to the surface of the polymerizedaminosilylated solid supports of the invention because of the highdensity of primary amines providing more points for attachment. Also, inthe case of biochips utilizing an enzyme or other biomolecules, the hostmacromolecules can be more stably attached to the polymerized layer onthe top of the solid support. Therefore, this invention improves thestability of biochips and thereto improving the manufacture and handlingprocesses.

The present invention will now be explained in more detail withreference to the following Examples. As will be realized, the inventionis capable of being modified in various obvious respects, all withoutdeparting from the invention. Accordingly, the drawings and descriptionare to be regarded as illustrative in nature, and not restrictive.However, these are to illustrate the present invention and the presentinvention is not limited thereto.

EXAMPLES Example 1 Aminosilylation and Polymerization of a Solid Support

A silica or glass substrate thoroughly washed and dried in vacuum ofapproximately 20 mTorr was immersed and incubated in the toluenesolution containing 10⁻³ M of (3-aminopropyl)diethoxymethylsilane undernitrogen at room temperature. Typically, the substrate is rectangular(10×30×1.5 mm for fused silica; 10×30×0.5 mm for silicon wafer;25×75×1.0 mm for glass slide) in shape. The fused silica isUV-transparent materials made of molten SiO₂, whereas silicon wafer ismade of pure silicon (Si). Because the surface of the latter has anaturally formed oxide layer in the air, the surface is practically thesame as that of the fused silica. However, it is not transparent toUV-vis light, the underlying bulk materials, Si, absorb the light. Aglass slide made of soda lime glass can be applied for the modification,and glass slides made of other materials such as borosilicate glass canalso be employed. Because of the simplicity in terms of the compositionand the regularity of the surface, more satisfactory results will beobtained with the fused silica and the silicon wafer.

After the aminosilylation reaction, the substrate was washed usingtoluene and baked at 120° C. for 30 minutes. The substrate was cooleddown to room temperature, and washed by sonication for 3 minutessequentially in toluene, mixture of toluene and methanol (volume ratioof 1:1), and then methanol.

The aminosilylated silica substrate was immersed in dichloromethanesolution (20 ml) including 0.2 ml of aziridine and a drop (about 20microliter) of acetic acid. The solution was heated to reflux at 80° C.for 24 hours under nitrogen.

The resulting substrate was thoroughly washed with a copious amount ofdichloromethane and sonicated in methanol three times. Finally, thewashed substrate was dried in vacuum at room temperature.

Example 2 Reaction with Protected Aziridine and Elimination of theProtecting Group

The same procedure as in Example 1 was used except that toluene wasemployed as the solvent for the reaction. In addition, a protectedaziridine derivative, benzyl 1-aziridinecarboxylate (N-Cbz aziridine),was used instead of aziridine for treating the primary amines on thesurface of the substrate.

The N-Cbz group was eliminated from the modified surface with thefollowing procedure. The silica substrate was immersed in neattrifluoroacetic acid and sonicated for 20 min at room temperature.Subsequently, the substrate was washed with a large amount of methanoland sonicated in methanol for 10 min three times.

Example 3 Aminosilylation of the Surface of a Silica Substrate Using(3-Aminopropyl) dimethylethoxysilane For a Lower Initial Density of thePrimary Amine Groups of a Substrate

The primary amines on the surface of a silica substrate and N-Cbzaziridine were reacted using the same method as in Example 1, exceptthat (3-aminopropyl)dimethylethoxysilane was used instead of(3-aminopropyl)diethoxymethylsilane for the aminosilylation step to forma lower initial density of primary amine groups on the surface of asubstrate.

Example 4 Confirmation of the Primary Amine Density and the Thickness ofthe Aminosilylated Layer Before and After Polymerization With Aziridine

The surface primary-amine density and thickness of the aminosilylatedlayer built on the silica substrate in Example 1 were measured beforetreating the solid support with aziridine. The thickness of theaminosilylated surface was measured with an optical ellipsometer, andthe surface density of the amine was measured with a method developed byMoon et al. The thickness of the aminosilylated layer was 8 Å and thesurface density of the functional groups was 3.5 amines/nm².

After the reaction with aziridine, the thickness of the film on thesilica substrate was measured again. The thickness of the film increasedincrementally with the reaction time and reached 36 Å in 24 hours.Therefore, it seems that the growth meets a saturation point where thepropagation rate decreases. At the saturation point, the measureddensity of the primary amine groups was 66 amines/nm². Beyond thispoint, the absolute density of the primary amine groups was constant.

Example 5 Confirmation of the Primary Amine Density and the Thickness ofa Film of the Hyperbranched Polymer on the Surface of a Solid Support

In accordance with the invention, the reaction between aziridine and theaminosilylated substrate goes through a ring-opening polymerization toform hyperbranched polymer, i.e., poly(ethyleneimine) on the supportsurface (FIG. 1). The polymerization can result in either a linear chainor a branched one, depending on whether the primary amine groups areconverted to secondary or tertiary amines upon reacting with aziridine.

A reliable way to judge whether the branching occurs is to approximatethe surface density of the end group, i.e., the primary amine. Theabsorbance at 284 nm owing to 4-nitrobenzaldimine occurs when theprimary amine is converted to the imine. By comparing absorbance beforeand after ring-opening polymerization, a relative approximation ofsurface density was determined for the silica substrate of Example 1following the procedure in Moon et al., Langmuir13,4305 (1997). As shownin FIG. 3, in case of Example 1, due to the increased number of primaryamines after the polymerization, the absorbance at 284 nm was sharplyhigher.

The absolute density of the amine groups was also determined byhydrolyzing the imine in a known amount of water following the procedureof Moon et al. It was found that the absolute density of the primaryamines increased dramatically from 3.5 amines/nm² to 66 amines/nm² in 24h as shown in Table 1. The observation definitely shows that branchingwas operative; otherwise a constant surface density would be obtainedregardless of the chain growth. It is noteworthy that the density didnot increase after 24 hours. Taking the slow but persistent increase ofthe thickness after 24 hours into consideration, branching remainsconstant beyond a particular stage.

TABLE 1 Contact Thickness Absolute density Stage angle(°) (Å) (number ofprimary amines/nm²) After 62(+2) 8 3.5 aminosilylation After 62(+2) 3666 polymerization

In fact, the growth accompanying the branching in solution appeared tostop after 3 or 4 cycles, which was attributed to steric hindrance.Therefore, the increase of the surface density should be less than16-times (2⁴) upon the branching because of the spatial restriction. Theobserved higher increase of the density (ca. 19-times) indicated thatthe length of the polymer chains varied. Polymer chains that areirregular in terms of length give a rougher surface (i.e., a more unevensurface morphology) and provide a higher surface density of primaryamine groups than that of a population of similar length polymer chains.

Example 6 Confirmation of the Surface Density of a Primary Amine and theThickness of a Polymer Film Constructed on the Surface Having LowerDensity of the Primary Amine

The same ring-opening polymerization reactions as in Example 1 andExample 2 were applied on silica substrates previously aminosilylatedwith (3-aminopropyl)dimethylethoxysilane to obtain an absolute primaryamine surface density of 1.5 amines/nm². The density of 1.5 amines/nm²is regarded as the low surface density relative to that of the substratefrom (3-aminopropyl)diethoxymethylsilane. The thickness of the film, asmeasured using the procedure of Moon et al., increased from 4 to 18 Åupon reaction in 24 hours. Also, the absolute surface density of theprimary amine group increased to 48 amines/nm². It seems that thesurface density approaches the one observed in either example 1 orexample 2 despite its low initial density. This phenomenon indicatesthat the final surface density is mainly controlled by the molecularvolume that is governed by the Van der Waals radii.

Example 7 Confirmation of the Stepwise Growth of the Polymer UsingProtected Aziridine

The aminosilylated substrate of Example 2 before and afterpolymerization with the N-Cbz aziridine was analyzed following theprocedure of Moon et al. The aminosilylated layer (8 Å, 3.5 amines/nm²)formed with (3-aminopropyl)diethoxymethylsilane was allowed to reactwith N-Cbz aziridine in refluxing toluene. In order to complete thereaction, a solvent with a higher boiling point, toluene was chosen.

After the reaction, the surface density of the primary amine groups forthe polymerized substrate of Example 2 was measured by derivatizing theintact primary amine groups with 4-nitrobenzaldehyde, hydrolyzingthus-formed imine by dipping the derivatized substrate in a known volumeof water, and subsequently measuring the absorbance at 284 nm.

As anticipated, the primary amine groups were not detected due to thelack of absorbance at 284 nm. In other words, all the primary amineswere bound to N-Cbz aziridine providing only the modified amines on thevery top surface of the film (FIG. 2). The polymerized aminosilylatedsubstrate of Example 2 exhibited an increased contact angle of 70° whichreflects the hydrophobic nature of the modified surface. Also, thethickness of the film increased to 16 Å from 8 Å which matches thecalculated value. Upon the deprotection in trifluoroacetic acid, thewater contact angle returned to the value of a pristine aminosilylatedlayer (60° ), and the thickness reduced to 12 Å. The physical propertiesof the surface indicate that the deprotection is as successful as in theliquid phase. It is worthwhile to note that the absolute density of theprimary amine groups (3.5 amines/nm²) did not increase at all. Theincreased reaction time did not enhance the density of the resultingsurface. Upon the subsequent reaction with N-Cbz aziridine, it wasobserved that the thickness of the layer increased again. After thefirst full cycle, the thickness increased to 12 Å and after the secondfull cycle, it increased to 18 Å. Therefore, the thickness increased by6 Å after each full cycle. It is believed that the steric congestion ofthe protecting group inhibits the molecular chain from branching (i.e.,inhibits the conversion of the primary amines to tertiary amines).

Example 8 Confirmation of the Surface Morphology in a Polymerized SolidSupport Using AFM (Atomic Force Microscope)

The surface structure of a silica substrate having poly(ethyleneimine)constructed from the ring-opening polymerization reaction betweenaminosilylated substrate and aziridine in accordance with Example 1 wassurveyed using AFM (atomic force microscope (2 um×2 um)). The heightdifference in a partial intersection of the surface of aminosilylated(non-polymerized) substrate was 4.61 nm (FIG. 4a) and that ofpolymerized substrate was 6.23 nm (FIG. 4). Also, the average heightdifference in the surface of the polymerized substrate was 11.5 nm andthat of aminosilylated substrate was 9.89 nm. The data shows that thesurface of polymerized substrate is slightly rougher than the surface ofthe aminosilylated substrate. However, the difference in the averageheight variation is insignificant and both the surface structures showthe smooth and similar morphologies in the images from Atomic ForceMicroscope(AFM) (FIG. 4). AFM is now an analytical instrument usedwidespread worldwide due to its superb resolution. There is no samplepreparation step to take images. Samples of a moderate size (about 2-3cm in length) can be directly analyzed.

While the present invention has been described in detail with thereference to the preferred embodiments, those skilled in the art willappreciate that the various modifications and substitutions can be madethereto without departing from the spirit and the scope of the presentinvention as set forth in the appended claims.

What is claimed is:
 1. A method for forming solid substrates having ahigh density of primary amine groups, comprising: introducingaminosilane groups onto a surface of a substrate to form anaminosilylated substrate having primary amine groups on the surface; andcontacting the surface of the aminosilylated substrate with a solutioncomprising aziridine or an aziridine derivative in the presence of anacid catalyst and a solvent thereby increasing the density of primaryamine groups on the surface of the substrate.
 2. The method according toclaim 1, wherein the aziridine derivative is a protected aziridine. 3.The method according to claim 2, wherein the protected aziridine isrepresented by the following formula 2:

wherein X is selected from the group consisting of hydrogen, nitro,alkoxy, and mixtures thereof.
 4. The method according to claim 2,wherein a linear chain without branching is formed on the surface of anaminosilylated substrate through a ring-opening polymerization betweenthe primary amines of the aminosilylated substrate and the protectedaziridine.
 5. The method according to claim 2, wherein a hyperbranchedpoly(ethyleneimine) is formed on the surface of the aminosilylatedsubstrate through a ring-opening polymerization between the primaryamines of the aminosilylated substrate and the aziridine.
 6. The methodaccording to claim 2, wherein contacting the surface of anaminosilylated substrate with the aziridine or the aziridine derivativecomprises: immersing the aminosilylated substrate in the solution; andheating the solution having the immersed substrate.
 7. The methodaccording to claim 6, wherein the method further comprises after heatingcontacting the substrate with trifluoroacetic acid thereby removing theprotective group from the protected aziridine.
 8. The method accordingto claim 1, wherein the substrate is a hydroxylated substrate.
 9. Themethod according to claim 8, wherein the hydroxylated substrate is asilicon wafer.
 10. The method of claim 8, wherein the hydroxylatedsubstrate is silica.
 11. The method according to claim 1, wherein thesolvent is an organic solvent.
 12. The method according to claim 11,wherein the organic solvent is selected from the group consisting ofdichloromethane, toluene, acetonitrile and mixtures thereof.
 13. Themethod according to claim 1, wherein the acid catalyst is selected fromthe group consisting of acetic acid, p-toluenesulfonic acid, andmixtures thereof.
 14. The method according to claim 1, wherein thedensity of primary amine groups on the surface of the substrate isincreased to greater than 3.5 amines/nm².
 15. The method according toclaim 14, wherein the density of primary amine groups is at least 10amines/nm².
 16. The method according to claim 15, wherein the density ofprimary amine groups is at least 48 amines/nm².
 17. A solid substratehaving high density of primary amine groups which comprises anaminosilylated substrate having a surface polymerized with aziridine oran aziridine derivative, wherein the surface of the substrate has adensity of primary amine groups greater than 3.5 amines/nm².
 18. Thesolid substrate according to claim 17, wherein the density of primaryamine groups is at least 10 amines/nm².
 19. The solid substrateaccording to claim 18, wherein the density of primary amine groups is atleast 48 amines/nm².